Automated microscopes such as confocal laser scanning microscopes and wide-field fluorescence microscopes are powerful imaging tools that are especially valuable for inspection of biological samples. One important component in a typical automated microscope is an autofocusing system. Ideally, the autofocusing system should be able to adjust focus for the microscope's optical system to obtain high-contrast images and should do so in a fast, accurate and reliable manner. However, very few existing autofocusing systems, if any, can meet such requirements in a cost-efficient way. Most known autofocusing systems fall into two categories: image-based or confocal-based, both of which have notable shortcomings.
An image-based autofocusing system may rely on an image-processing algorithm to find a best focus for a microscope. Such an image-based autofocusing system may cause a main optical detector in the microscope to acquire a number of images while an objective lens is moved within its focus range, so that each image corresponds to a different focal position. The contrast of each image is then analyzed and evaluated by applying the image-processing algorithm to the image data. The best focal position is identified as one that corresponds to the strongest contrast in the acquired images. Although such a image-based autofocusing system is usually simple, reliable and inexpensive, it is relatively slow since multiple images have to be acquired and then processed with the complex contrast-evaluation algorithm. In addition, this type of image-based autofocusing cannot be performed during image acquisition (i.e., operate in a “tracking mode”) because the main optical detector would not be available for autofocusing operations.
An image-based autofocusing system may also rely on a separate set of hardware (e.g., a separate optical detector and separate optical components) that operates somewhat independently from the microscope's main image acquisition system. This type of autofocusing system is also referred to as a hardware-based system.
FIG. 1 shows a prior art microscope 100 equipped with a hardware-based autofocusing system 120. A main image acquisition system in the microscope 100 comprises a main charge coupled device (CCD) detector 102, a main optical path 104 that comprises an objective lens 110 and other optical elements, a main illumination light source 106, and an object stage 108. The main illumination light source 106 generates a light beam 10 (typically a laser) that is directed, via the main optical path 104, to the object stage 108 to illuminate a sample thereon. An image of the sample is then detected by the main CCD detector 102.
In addition to and independent from the main image acquisition system, the hardware-based autofocusing system 120 provides a secondary image acquisition system that includes a secondary CCD detector 122, a secondary optical path 124 and an autofocus light source 126. The autofocus light source 126 typically generates an autofocus light beam 20 having a different wavelength from the light beam 10 generated by the main illumination light source 106. The autofocus light beam 20 is coupled into the main optical path 104 to illuminate the object stage 108. A resulting image is then detected by the secondary CCD detector 122. A microcontroller (or microprocessor) 128 is available to analyze the autofocus images acquired by the secondary CCD detector 122. The microcontroller 128 is also coupled to a motion control unit 112 that adjusts a relative position of the object stage 108 with respect to the objective lens 110. By coordinating the movement of the object stage 108 and the acquisition and analysis of autofocus images, the microcontroller 128 may quickly identify an optimal focal position that produces the strongest contrast in the autofocus image. It should be noted that a hardware-based autofocusing system, such as the system 120, may also be adapted for confocal-based autofocusing, wherein a physical confocal aperture (not shown) is used and, instead of analyzing entire images, light intensity through the confocal aperture may be analyzed for focus adjustments.
With a secondary image acquisition system dedicated to autofocusing, a hardware-based autofocusing system can zero in on the best focus fairly fast and may also be able to perform focus adjustment when the microscope's main image acquisition system is busy acquiring sample images. However, the dedicated autofocusing hardware also substantially increases the overall complexity and cost of the microscope. Furthermore, a hardware-based autofocusing system is typically incapable of flexible adaptation to different imaging modes the microscope operates in.
Apart from confocal-based autofocusing, there is another type of non-image-based autofocusing approach known as position-sensitive autofocusing. Position-sensitive autofocusing can be used for tracking-mode focus adjustments, and is widely used in optical drives such as compact disk (CD) drives and digital versatile disk (DVD) drives. However, it is very difficult to implement position-sensitive autofocusing in an imaging system with multiple objective lenses.